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Faris A Alkhilaiwi,1,2 Sana A Fadil,1,2 Fadwa A Aljoud,2,3 Ahmed R Yonbawi,1 Abrar Ashi,2,4 Rawan H Hareeri,5 Sherin Bakhashab,6 Ahmad M Alamri,7,8 Ahmed Y Albikairi,1 Lamiaa A Shaala,9,10 Ali A El-Gamal,11,12 Diaa T Youssef1,10,13 1Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah, 21589, Saudi Arabia; 2Regenerative Medicine Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, 21589, Saudi Arabia; 3Scientific Research Center, Dar Al-Hekma University, Jeddah, 22246, Saudi Arabia; 4Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia; 5Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah, 21589, Saudi Arabia; 6Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, 21589, Saudi Arabia; 7Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, 61481, Saudi Arabia; 8Cancer Research Unit, King Khalid University, Abha, 61481, Saudi Arabia; 9Suez Canal University Hospital, Suez Canal University, Ismailia, 41522, Egypt; 10Natural Products Unit, King Fahad Medical Research Center, King Abdulaziz University, Jeddah, 21589, Saudi Arabia; 11Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, 11451, Saudi Arabia; 12Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, El-Mansoura, 35516, Egypt; 13Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia, 41523, EgyptCorrespondence: Faris A Alkhilaiwi; Diaa T Youssef, Email Faalkhilaiwi@kau.edu.sa; dyoussef@kau.edu.saBackground: Breast cancer is a leading cause of death and one of the most common fatal medical conditions in the world. Chemical compounds of various types have been identified in the Red Sea marine sponge Xestospongia testudinaria, including sterol esters, sterols, indole alkaloids, and brominated polyunsaturated fatty acids. These compounds have demonstrated promising biological features, which in cludes anti-inflammatory, cancer preventive, and antioxidant capacities.Methods: The cytotoxic potential of Xestospongia testudinaria was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and morphological alterations in MCF-7 cell line. Furthermore, the flow cytometry was also utilized to assess apoptosis and identify changes in the cell cycle; besides, cell migration was assessed by scratch wound-healing assay.Results: A significant dose-dependent decrease in the percentage of MCF-7 cell viability was observed with IC50 39.8 ug/mL. Functional studies were performed on MCF-7 to show that Xestospongia testudinaria raises apoptotic cell death and induces growth arrest at the G1/G0 while inhibiting cell migration in scratch assay.Conclusion: These results demonstrated that Xestospongia testudinaria extract has an inhibitory effect on breast cancer cells proliferation, migration and induce apoptosis. Thus, it holds great promise as a potential treatment for breast cancer. Keywords: Red Sea sponge, Xestospongia testudinaria, breast cancer cell lines, cytotoxic activity

The present study focuses on the evaluation of the cytotoxicity and antiproliferative activities of the organic extracts of 70 fungal strains associated with twelve Red Sea marine invertebrates. The fungal strains were obtained 10 sponges, one tunicate and one soft coral. Three different media including Sabouraud dextrose agar, malt extract agar and Czapek-Dox agar were used for the purification of the fungal isolates. The purified fungal isolates were cultured in their corresponding media (Sabouraud dextrose broth, Malt extract broth and Czapek-Dox broth) on shaker for 14 days at 26° C. After that, the cultures were lyophilized and the dried cultures were extracted with methanol. The methanolic extracts of these cultures were evaluated for their in vitro cytotoxicity and antiproliferative activities against three human cancer cell lines including breast adenocarcinoma (MCF-7), liver hepatocellular carcinoma (HepG2) and colorectal carcinoma (HCT-116). Nine extracts displayed potent and selective activity against MCF-7 with IC 50 4.96-8.28μ g/mL without any significant effect on the other two cell lines. In addition, six extracts showed strong and selective activity against MCF-7 with IC 50 11.37-15.53μ g/mL. On the other hand, most of the fungal extracts were inactive or weakly active against HepG2 and HCT-116.

Two new antimicrobial fatty acid esters, penicilloitins A and B (1 and 2), along with α-cyclopiazonic acid (3), tryptamine (4) and indole-3-carbaldehyde (5), were isolated from mycelial extracts and the culture broth of a marine endophytic Penicillium species and their structures established by spectroscopic techniques including 1D (1H NMR and 13C NMR) and 2D NMR (COSY, multiplicity-edited HSQC, and HMBC) as well as HRESIMS. Of these compounds, 1, 3 and 4 displayed modest antimicrobial activity.

As a part of our ongoing effort to allocate marine microbial bioactive leads, a tunicate-derived actinomycete, Streptomyces sp. Did-27, was investigated. Three new 2(1H)-pyrazinones derivatives, (S)-6-(sec-butyl)-3-isopropylpyrazin-2(1H)-one (1), (S)-3-(sec-butyl)-6-isopropylpyrazin-2(1H)-one (2) and (S)-6-(sec-butyl)-3-isobutylpyrazin-2(1H)-one (3), together with the known (1H)-pyrazinones analogues deoxymutaaspergillic acid (4), 3,6-diisobutyl-2(1H)-pyrazinone (5) and 3,6-di-sec-butyl-2(1H)-pyrazinone (6), and the diketopiperazine alkaloids cyclo(6-OH-d-Pro-l-Phe) (7), bacillusamide B (8), cyclo(l-Pro-l-Leu) and cyclo(l-Pro-l-Ile) (10) were isolated from this strain. The structures of the compounds were determined by study of their one- and two-dimensional NMR spectra as well as high-resolution mass spectral determinations. Compound 4 was reported previously as a synthetic product, while compound 6 was reported as 2-hydroxy-3,6-di-sec-butylpyrazine. Herein, we report the complete NMR data for compounds 4 and 6. The compounds were evaluated for their cytotoxic activities against three cell lines. Compound 5 showed potent and selective activity against HCT-116 cell line with IC50 of 1.5 μg/mL, while 1-10 showed variable cytotoxic activities against these cancer cell lines. These results provide further understanding about the chemistry and bioactivities of the alkylated 2(1H)-pyrazinone derivatives.

As a part of our continuing work to find out bioactive lead molecules from marine invertebrates, the CHCl₃ fraction of the organic extract of the Red Sea sponge Theonella mirabilis showed cytotoxic activity in our primary screen. Bioassay-guided purification of the active fractions of the sponge's extract resulted in the isolation of two new glycerides, mirabolides A and B (1 and 2), together with the reported 4-methylene sterols, conicasterol (3) and swinhosterol B (4). The structures of the compounds were assigned by interpretation of their 1D (¹H, (13)C), 2D (COSY, HSQC, HMBC, ROESY) NMR spectral data and high-resolution mass determinations. Compounds 1-4 displayed marked cytotoxic activity against human breast adenocarcinoma cell line (MCF-7) with IC50 values of 16.4, 5.18, 6.23 and 3.0 μg/mL, respectively, compared to 5.4 μg/mL observed by doxorubicin as reference drug.

Bioassay-guided fractionation of the organic extract of the Red Sea sponge Xestospongia testudinaria led to the isolation of 13 compounds including two new sterol esters, xestosterol palmitate (2) and xestosterol ester of l6'-bromo-(7'E,11'E,l5'E)-hexadeca-7',11',l5'-triene-5',13'-diynoic acid (4), together with eleven known compounds: xestosterol (1), xestosterol ester of 18'-bromooctadeca-7'E,9'E-diene-7',15'-diynoic acid (3), and the brominated acetylenic fatty acid derivatives, (5E,11E,15E,19E)-20-bromoeicosa-5,11,15,19-tetraene-9,17-diynoic acid (5), 18,18-dibromo-(9E)-octadeca-9,17-diene-5,7-diynoic acid (6), 18-bromooctadeca-(9E,17E)-diene-7,15-diynoic acid (7), 18-bromooctadeca-(9E,13E,17E)-triene-7,15-diynoic acid (8), l6-bromo (7E,11E,l5E)hexadeca-7,11,l5-triene-5,13-diynoic acid (9), 2-methylmaleimide-5-oxime (10), maleimide-5-oxime (11), tetillapyrone (12), and nortetillapyrone (13). The chemical structures of the isolated compounds were accomplished using one- and two-dimensional NMR, infrared and high-resolution electron impact mass spectroscopy (1D, 2D NMR, IR and HREIMS), and by comparison with the data of the known compounds. The total alcoholic and n-hexane extracts showed remarkable cytotoxic activity against human cervical cancer (HeLa), human hepatocellular carcinoma (HepG-2), and human medulloblastoma (Daoy) cancer cell lines. Interestingly, the dibrominated C18-acetylenic fatty acid (6) exhibited the most potent growth inhibitory activity against these cancer cell lines followed by Compounds 7 and 9. Apparently, the dibromination of the terminal olefinic moiety has an enhanced effect on the cytotoxic activity.

In the course of our ongoing efforts to identify marine-derived bioactive compounds, the marine cyanobacterium Moorea producens was investigated. The organic extract of the Red Sea cyanobacterium afforded one new cerebroside, mooreaside A (1), two new nucleoside derivatives, 3-acetyl-2'-deoxyuridine (2) and 3-phenylethyl-2'-deoxyuridine (3), along with the previously reported compounds thymidine (4) and 2,3-dihydroxypropyl heptacosanoate (5). The structures of the compounds were determined by different spectroscopic studies (UV, IR, 1D, 2D NMR, and HRESIMS), as well as comparison with the literature data. Compounds 1-5 showed variable cytotoxic activity against three cancer cell lines.

In the course of our continuing efforts to identify bioactive secondary metabolites from Red Sea marine invertebrates, we have investigated the sponge Hemimycale arabica. The antimicrobial fraction of an organic extract of the sponge afforded two new hydantoin alkaloids, hemimycalins A and B (2 and 3), together with the previously reported compound (Z)-5-(4-hydroxybenzylidene)imidazolidine-2,4-dione (1). The structures of the compounds were determined by extensive 1D and 2D NMR (COSY, HSQC and HMBC) studies and high-resolution mass spectral determinations. Hemimycalins A (2) and B (3) represent the first examples of the natural N-alkylated hydantoins from the sponge Hemimycale arabica. Compounds 1-3 displayed variable antimicrobial activities against E. coli, S. aureus, and C. albicans. In addition, compound 1 displayed moderate antiproliferative activity against the human cervical carcinoma (HeLa) cell line. These findings provide further insight into the chemical diversity as well as the biological activity of this class of compounds.

Marine sponges are found to be a rich source of bioactive compounds which show a wide range of biological activities including antiviral, antibacterial, and anti-inflammatory activities. This study aimed to investigate the possible anti-inflammatory, antioxidant and immunomodulator effects of the methanolic extract of the Red Sea marine sponge Xestospongia testudinaria. The chemical composition of the Xestospongia testudinaria methanolic extract was determined using Gas chromatography-mass spectroscopy (GC-MS) analysis. DPPH (2, 2-diphenyl-1-picryl-hydrazyl) was measured to assess the antioxidant activity of the sponge extract. Carrageenan-induced rat hind paw edema was adopted in this study. Six groups of rats were used: group1: Control, group 2: Carrageenan, group 3: indomethacin (10 mg/kg), group 4-6: Xestospongia testudinaria methanolic extract (25, 50, and 100 mg/kg). Evaluation of the anti-inflammatory activity was performed by both calculating the percentage increase in paw weight and hisopathologically. Assessment of the antioxidant and immunomodulatory activity was performed. GC-MS analysis revealed that there were 41 different compounds present in the methanolic extract. Sponge extract exhibited antioxidant activity against DPPH free radicals. Xestospongia testudinaria methanolic extract (100 mg/kg) significantly decreased % increase in paw weight measured at 1, 2, 3 and 4 h after carrageenan injection. Histopathologically, the extract caused a marked decrease in the capillary congestion and inflammatory cells infiltrate. The extract decreased paw malondialdehyde (MDA) and nitric oxide (NO) and increased the reduced glutathione (GSH), glutathione peroxidase (GPx), and catalase (CAT) activity. It also decreased the inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-1 β(IL-1β) and IL-6. The results of this study demonstrated the anti-inflammatory, antioxidant, and immunomodulatory effects of the methanolic extract of the Red Sea sponge Xestospongia testudinaria (100 mg/kg).

Our previous studies showed that several sipholane triterpenes, sipholenol A, sipholenone E, sipholenol L and siphonellinol D, have potent reversal effect for multidrug resistance (MDR) in cancer cells that overexpressed P-glycoprotein (P-gp/ABCB1). Through comparison of cytotoxicity towards sensitive and multi-drug resistant cell lines, we identified that the semisynthetic esters sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate potently reversed P-gp-mediated MDR but had no effect on MRP1/ABCC1 and BCRP/ABCG2-mediated MDR. The results from [3H]-paclitaxel accumulation and efflux studies suggested that these two triterpenoids were able to increase the intracellular accumulation of paclitaxel by inhibiting its active efflux. In addition, western blot analysis revealed that these two compounds did not alter the expression levels of P-gp when treated up to 72 h. These sipholenol derivatives also stimulated the ATPase activity of P-gp membranes, which suggested that they might be substrates of P-gp. Moreover, in silico molecular docking studies revealed the virtual binding modes of these two compounds into human homology model of P-gp. In conclusion, sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate efficiently inhibit the P-gp and may represent potential reversal agents for the treatment of multidrug resistant cancers.

As a part of our continuing interest in identifying anticancer drug leads from natural sources, we have investigated the n-BuOH fraction of the root bark of Calotropis procera (Ait) R. Br. Seven new oxypregnane oligoglycosides: calotroposides H-N (1-7) were isolated and identified. Their structures were established on the basis of 1D and 2D NMR studies, HRMS, and GCMS spectral data. The in vitro growth inhibitory activity of the n-BuOH fraction and compounds 1-7 was evaluated against A549 non-small cell lung cancer (NSCLC), U373 glioblastoma (GBM), and PC-3 prostate cancer cell lines. Compounds 4 and 6 showed subnanomolar growth inhibition activity with IC50 ranging from 0.5 to 0.7μM against U373 glioblastoma (GBM) and PC-3 prostate cancer cell lines. These results provide further insight into the chemical diversity and biological activities of this class of compounds., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In the course of our continuous interest in identifying bioactive compounds from marine microbes, we have investigated a tunicate-derived fungus, Penicillium sp. CYE-87. A new compound with the 1,4-diazepane skeleton, terretrione D (2), together with the known compounds, methyl-2-([2-(1H-indol-3-yl)ethyl]carbamoyl)acetate (1), tryptamine (3), indole-3-carbaldehyde (4), 3,6-diisobutylpyrazin-2(1H)-one (5) and terretrione C (6), were isolated from Penicillium sp. CYE-87. The structures of the isolated compounds were established by spectral analysis, including 1D (1H, 13C) and 2D (COSY, multiplicity edited-HSQC and HMBC) NMR and HRESIMS, as well as comparison of their NMR data with those in the literature. The compounds were evaluated for their antimigratory activity against the human breast cancer cell line (MDA-MB-231) and their antiproliferation activity against HeLa cells. Compounds 2 and 6 showed significant antimigratory activity against MDA-MB-231, as well as antifungal activity against C. albicans.

In a continuation of our efforts to identify bioactive compounds from Red Sea Verongid sponges, the organic extract of the sponge Suberea species afforded seven compounds including two new dibrominated alkaloids, subereamollines C and D (1 and 2), together with the known compounds aerothionin (3), homoaerothionin (4), aeroplysinin-1 (5), aeroplysinin-2 (6) and a revised subereaphenol C (7) as ethyl 2-(2,4-dibromo-3,6-dihydroxyphenyl)acetate. The structures of the isolated compounds were assigned by different spectral data including optical rotations, 1D (1H and 13C) and 2D (COSY, multiplicity-edited HSQC, and HMBC) NMR and high-resolution mass spectroscopy. Aerothionin (3) and subereaphenol C (7) displayed potent cytotoxic activity against HeLa cell line with IC50 values of 29 and 13.3 µM, respectively. In addition, aeroplysinin-2 (6) showed potent antimigratory activity against the human breast cancer cell line MDA-MB-231 with IC50 of 18 µM. Subereamollines C and D are new congeners of the previously reported compounds subereamollines A and B with methyl ester functionalities on the side chain. These findings provide further insight into the biosynthetic capabilities of members of the genus Suberea and the chemical diversity as well as the biological activity of these compounds.

Chemical investigation of the organic extract of a Red Sea strain of the cyanobacterium Moorea producens has afforded 2,3-seco-2,3-dioxo-lyngbyatoxin A (1). Five known compounds including lyngbyatoxin A (2), majusculamides A and B (3 and 4), aplysiatoxin (5) and debromoaplysiatoxin (6) were also isolated. Their structures were elucidated by using HR-FAB-MS, 1D and 2D NMR analyses. The compounds were evaluated for antiproliferative activity against HeLa cancer cells. Lyngbyatoxin A (2) showed potent activity, with an IC50 of 9.2 nM, while 5 and 6 displayed modest activity with IC50 values of 13.3 and 3.03 μM, respectively. In contrast, compounds 1, 3 and 4 were inactive, with IC50 values greater than 50 μM. The lack of cytotoxicity for 2,3-seco-2,3-dioxo-lyngbyatoxin A (1) demonstrates that the indole moiety in lyngbyatoxin (2) is essential for its cytotoxicity, and suggests that detoxification of 2 may be carried out by biological oxidation of the indole moiety to yield 1.

In continuation of our ongoing efforts to identify bioactive compounds from Red Sea marine organisms, a new collection of the ascidian Didemnum species was investigated. Chromatographic fractionation and HPLC purification of the CH2Cl2 fraction of an organic extract of the ascidian resulted in the identification of two new spiroketals, didemnaketals F (1) and G (2). The structure determination of the compounds was completed by extensive study of 1D (1H, 13C, and DEPT) and 2D (COSY, HSQC, and HMBC) NMR experiments in addition to high-resolution mass spectral data. Didemnaketal F (1) and G (2) differ from the previously reported compounds of this class by the lack the terminal methyl ester at C-1 and the methyl functionality at C-2. Instead, 1 and 2 possess a methyl ketone moiety instead of the terminal ester. Furthermore, didemnaketal F possesses a disubstituted double bond between C-2 and C-3, while the double bond was replaced by a secondary alcohol at C-3 in didemnaketal G. In addition, they possess the unique spiroketal/hemiketal functionality which was previously reported in didemnaketal E. Didemnaketals F (1) and G (2) displayed moderate activity against HeLa cells with of IC50s of 49.9 and 14.0 µM, respectively. In addition, didemnaketal F (1) displayed potent antimicrobial activity against E. coli and C. albicans. These findings provide further insight into the biosynthetic capabilities of this ascidian and the chemical diversity as well as the biological activity of this class of compounds.

In our search for bioactive metabolites from marine organisms, we have investigated the polar fraction of the organic extract of the Red Sea sponge Theonella swinhoei. Successive chromatographic separations and final HPLC purification of the potent antifungal fraction afforded a new bicyclic glycopeptide, theonellamide G. The structure of the peptide was determined using extensive 1D and 2D NMR and high-resolution mass spectral determinations. The absolute configuration of theonellamide G was determined by chemical degradation and 2D NMR spectroscopy. Theonellamide G showed potent antifungal activity towards wild and amphotericin B-resistant strains of Candida albicans with IC₅₀ of 4.49 and 2.0 μM, respectively. Additionally, it displayed cytotoxic activity against the human colon adenocarcinoma cell line (HCT-16) with IC₅₀ of 6.0 μM. These findings provide further insight into the chemical diversity and biological activities of this class of compounds.

Phytochemical investigation of the cytotoxic fractions of fresh bulbs of Pancratium maritimum L. led to the isolation and structure identification of two new compounds, pancricin (1) and pancrichromone (4), together with four known compounds, including 2,4-dihydroxy-6-methoxy-3-methyl acetophenone (2), 5-formylfurfuryl acetate (3), 7-beta-D-glucosyloxy-5-hydroxy-2-methylchromone (5), and ethyl-beta-D-glucopyranoside (6). Their structures were established on the basis of 1D and 2D NMR spectroscopy (1H, 13C, COSY, HSQC, and HMBC), as well as HR mass spectral analyses. The compounds were evaluated for their antimigratory and antiproliferative activities against the highly metastatic human prostate cancer cell line (PC-3M). Compound 5 was the most active compound displaying good activity in the proliferation assay comparable to that of the positive control 4-hydroxyphenylmethylene hydantoin, while it displayed only weak antimigratory activity compared to the positive control 4-ethylmercaptophenylmethylene hydantoin.

Chemical investigation of the Red Sea sponge Mycale euplectellioides afforded two new compounds; hexacosa-(6Z,10Z)-dienoic acid methyl ester (1) and hexacosa-(6Z,10Z)-dienoic acid (2), along with two known compounds: icosa-(8Z,11Z)-dienoic acid methyl ester (3) and β-sitosterol (4). The structures were elucidated by the interpretation of their spectral data. The total methanol extract (TME) of the sponge exhibited potent antimicrobial activity against the different strains at a concentration of 100 mg/mL. All tested fractions did not exhibit any activity against Serratia marcescens and tested fungal strains. The TME and different fractions displayed anti-inflammatory and antipyretic activities at doses of 100 and 200 mg/kg compared with indomethacin (8 mg). The TME exhibited a remarkable hepato-protective effect in CCl4-induced liver damage compared with silymarin. Furthermore, compounds 1 and 2 displayed weak activity against A549 non-small cell lung cancer, the U373 glioblastoma and the PC-3 prostate cancer cell lines.

Two new glycerides, didemnacerides A (1) and B (2), together with three known sterols, 24-ethyl-25-hydroxycholesterol (3), cholest-6-en-3,5,8-triol (4) and cholestane-3β,5α,6β-26-tetrol (5), were isolated from the Red Sea ascidian Didemnum sp. Their structures were elucidated by using extensive 1D ((1)H, (13)C) and 2D ((1)H-(1)H COSY, HSQC and HMBC) NMR studies and mass spectroscopic data (GC-MS and HR-MS) as well as alkaline hydrolysis followed by GC-MS and NMR spectral analyses of the fatty acid methyl esters. This is the first report of compounds 3-5 from the Red Sea ascidian Didemnum species.

Recent studies have demonstrated that marine sponges and their active constituents exhibited several potential medical applications. This study aimed to evaluate the possible hepatoprotective role as well as the antioxidant effect of the Red Sea Suberea mollis sponge extract (SMSE) on carbon tetrachloride- (CCl4-) induced acute liver injury in rats. In vitro antioxidant activity of SMSE was evaluated by 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay. Rats were orally administered three different concentrations (100, 200, and 400 mg/kg) of SMSE and silymarin (100 mg/kg) along with CCl4 (1 mL/kg, i.p., every 72 hr) for 14 days. Plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bilirubin were measured. Hepatic malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide (NO), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were also measured. Liver specimens were histopathologically examined. SMSE showed strong scavenging activity against free radicals in DPPH assay. SMSE significantly reduced liver enzyme activities. Moreover, SMSE significantly reduced hepatic MDA formation. In addition, SMSE restored GSH, NO, SOD, GPx, and CAT. The histopathological results confirmed these findings. The results of this study suggested a potent protective effect of the SMSE against CCl4-induced hepatic injury. This may be due to its antioxidant and radical scavenging activity.

The microbial transformation of vitamin D3 (1) by the fungi Candida maltosa R42 and Botrytis allii NRRL 2502 was investigated. Incubation of compound 1 with C. maltosa R42 and B. allii NRRL 2502 produced the same three more polar metabolites in small yields. The main metabolite was identified as 1α-hydroxyvitamin D3 (2). This biotransformation has utility as a possible tool for the production of 1α-hydroxyvitamin D3 from the readily available vitamin D3 for patients with compromised kidney function.

One new compound, (2S)-1-O-(Z)-tetracos-6-enoate glycerol (1) named urgineaglyceride A, along with six known compounds, 3,5,7,3',5'-pentahydroxydihydroflavonol (2), stigmasterol (3), (25S)-5β-furostane-3β-22α-26-triol (4), scillaridin A (5), (2S)-(+)-2-hydroxynaringenin-4'-O-β-D-glucopyranoside (6) and quercetin-3'-O-β-D-glucopyranoside (7), were isolated from the EtOAc fraction of Drimia maritima (L.) Stearn bulbs. Their structures were secured based on their IR, UV, 1D and 2D NMR data, in addition to HR-MS data and comparison with the literature data. The isolated compounds were evaluated for their in vitro growth inhibitory activity against A549 non-small cell lung cancer (NSCLC), U373 glioblastoma (GBM) and PC-3 prostate cancer cell lines. Compounds 2 and 3 displayed variable activities against the tested cancer cell lines. Compound 2 was a selective inhibitor of the NSCLC cell line with an IC₅₀ of 2.3 μM, whereas 3 was selective against GBM with IC₅₀ of 0.5 μM and against PC-3 with 2.0 μM.

We have studied the ethyl acetate fraction of the methanolic extract of the root barks of Calotropis procera (Asclepiadaceae) from Egypt. Bioassay-directed fractionation and final purification of the extract resulted in the identification of a new cardenolide glycoside named proceraside A (1) together with two known compounds, frugoside (2) and calotropin (3). Their structures were elucidated by extensive NMR studies and mass spectrometric data. The in vitro cytotoxicity of the isolated compounds was evaluated against A549 non-small cell lung cancer, U373 glioblastoma and PC-3 prostate cancer cell lines. They showed potent activity against the tested cancer cell lines with IC50 ranging from 0.005 to 0.3 μg/mL. Cisplatin was used as positive control.

Chemical investigation of the Red Sea sponge Dragmacidon coccinea led to the isolation of a new nucleoside, dragmacidoside (1), along with eight known compounds: adenosine (2), inosine (3), deoxycytidine (4), methyl-α-d-glucopyranoside (5), clionasterol (6), stigmasterol (7), campesterol (8) and brassicasterol (9). The compounds were isolated from chloroform and ethyl acetate fractions of the methanolic extract of the sponge, and their structures were established based on various spectroscopic data including MS, 1D and 2D NMR (COSY, HSQC and HMBC). Biological testing revealed that the chloroform fraction possesses significant anti-inflammatory activity in the carrageenan-induced hind paw oedema in rats.

As a part of ongoing search efforts for the discovery of anticancer lead entities from natural sources, bulbs and flowers of the amaryllidaceous plant Pancratium maritimum have been investigated. Fractionation of the extracts of the fresh flowers and bulbs of P. maritimum led to the isolation of four new alkaloids, namely pancrimatines A (1) and B (2), norismine (3), and pancrimatine C (4), together with the previously reported N-methyl-8,9-methylenedioxy-6-phenanthridone (5), trispheridine (6), and N-methyl-8,9-methylenedioxyphenanthridine (7). The structures of these alkaloids were established on the basis of extensive 1D and 2D NMR and high-resolution mass spectral analyses as well as comparison with the literature. Compounds 2 and 7 showed antiproliferative and antimigratory activity against the highly metastatic human prostate cancer cell line PC-3 cells without cytotoxicity. The phenanthridine alkaloid class was identified as having potential for use to control prostate cancer proliferation and migration., (Georg Thieme Verlag KG Stuttgart · New York.)

Cultivation of the marine cyanobacterium Moorea producens, collected from the Nabq Mangroves in the Gulf of Aqaba (Red Sea), led to the isolation of new apratoxin analogues apratoxin H (1) and apratoxin A sulfoxide (2), together with the known apratoxins A-C, lyngbyabellin B, and hectochlorin. The absolute configuration of these new potent cytotoxins was determined by chemical degradation, MS, NMR, and CD spectroscopy. Apratoxin H (1) contains pipecolic acid in place of the proline residue present in apratoxin A, expanding the known suite of naturally occurring analogues that display amino acid substitutions within the final module of the apratoxin biosynthetic pathway. The oxidation site of apratoxin A sulfoxide (2) was deduced from MS fragmentation patterns and IR data, and 2 could not be generated experimentally by oxidation of apratoxin A. The cytotoxicity of 1 and 2 to human NCI-H460 lung cancer cells (IC₅₀ = 3.4 and 89.9 nM, respectively) provides further insight into the structure-activity relationships in the apratoxin series. Phylogenetic analysis of the apratoxin-producing cyanobacterial strains belonging to the genus Moorea, coupled with the recently annotated apratoxin biosynthetic pathway, supports the notion that apratoxin production and structural diversity may be specific to their geographical niche.

In continuation of our search for drug leads from Red Sea sponges we have investigated the ethyl acetate fraction of the organic extract of the Red Sea sponge Hyrtios species. Bioassay-directed fractionation of the active fraction resulted into the identification of three new alkaloids, hyrtioerectines D-F (1-3). Hyrtioerectines D-F belong to the rare marine alkaloids in which the indole and β-carboline fragments of the molecule are linked through C-3/C-3 of both moieties. The structures of the isolated compounds were established based on different spectroscopic data including UV, IR, 1D and 2D NMR (COSY, HSQC, and HMBC) and high-resolution mass spectral studies. The antimicrobial activity against several pathogens and the free radical scavenging activity of the compounds using DPPH reagent were evaluated. In addition, the growth inhibitory activity of the compounds against three cancer cell lines was also evaluated. Hyrtioerectines D-F (1-3) displayed variable antimicrobial, free radical scavenging and cancer growth inhibition activities. Generally, compounds 1 and 3 were more active than compound 2.

Investigation of the marine sponge Dysidea avara, family Dysideidae, afforded a new sesquiterpene (-)-N-methylmelemeleone-A (5), in addition to four known sesquiterpenes (+)-avarol (1), (+)-avarone (2), (-)-3'-methylaminoavarone (3) and (-)-4'-methylaminoavarone (4). The structure elucidation of compound 5 was based on 1D and 2D NMR spectroscopic, and HR-MS studies, as well as by comparison with the literature. Cytotoxicity, proteinkinase inhibition, inhibition of NFkB-activity and insecticidal activity were evaluated for the isolated compounds.

A new collection of several Red Sea sponges was investigated for the discovery of potential breast cancer migration inhibitors. Extracts of the Verongid sponges Pseudoceratina arabica and Suberea mollis were selected. Bioassay-directed fractionation of both sponges, using the wound-healing assay, resulted into the isolation of several new and known brominated alkaloids. Active fractions of the sponge Pseudoceratina arabica afforded five new alkaloids, ceratinines A-E (2-6), together with the known alkaloids moloka'iamine (1), hydroxymoloka'iamine (7) and moloka'iakitamide (8). The active fraction of the sponge Suberea mollis afforded the three known alkaloids subereamolline A (9), aerothionin (10) and homoaerothionin (11). Ceratinine B (3) possesses an unprecedented 5,7-dibrominated dihydroindole moiety with an epoxy ring on the side chain of a fully substituted aromatic moiety. Ceratinines D (5) and E (6) possess a terminal formamide moiety at the ethylamine side chain. Subereamolline A (9) potently inhibited the migration and invasion of the highly metastatic human breast cancer cells MDA-MB-231 at the nanomolar doses. Subereamolline A and related brominated alkaloids are novel scaffolds appropriate for further future use for the control of metastatic breast cancer.

Two new grassypeptolides and a lyngbyastatin analogue, together with the known dolastatin 12, have been isolated from field collections and laboratory cultures of the marine cyanobacterium Leptolyngbya sp. collected from the SS Thistlegorm shipwreck in the Red Sea. The overall stereostructures of grassypeptolides D (1) and E (2) and Ibu-epidemethoxylyngbyastatin 3 (3) were determined by a combination of 1D and 2D NMR experiments, MS analysis, Marfey's methodology, and HPLC-MS. Compounds 1 and 2 contain 2-methyl-3-aminobutyric acid and 2-aminobutyric acid, while biosynthetically distinct 3 contains 3-amino-2-methylhexanoic acid and the β-keto amino acid 4-amino-2,2-dimethyl-3-oxopentanoic acid (Ibu). Grassypeptolides D (1) and E (2) showed significant cytotoxicity to HeLa (IC₅₀ = 335 and 192 nM, respectively) and mouse neuro-2a blastoma cells (IC₅₀ = 599 and 407 nM, respectively), in contrast to Ibu-epidemethoxylyngbyastatin 3 (neuro-2a cells, IC₅₀ > 10 μM) and dolastatin 12 (neuro-2a cells, IC₅₀ > 1 μM).

Sarcophine (1) is a bioactive cembranoid diterpene isolated from the Red Sea soft coral Sarcophyton glaucum. Previous semisynthesis attempts resulted in decreased or complete loss of 1's anticancer activity. Sarcophine and analogues showed antimigratory activity against breast and prostate cancer cell lines. This encouraged further semisynthestic optimizations to improve its activity and establish a preliminary structure-activity relationship. Eight new and five known semisynthetic analogues were generated. These compounds were evaluated for their ability to inhibit growth, proliferation, and migration of the prostate and breast metastatic cancer cell lines PC-3 and MDA-MB-231, respectively. Most analogues exhibited enhanced antimigratory activity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Investigation of a new collection of the Red Sea sponge Suberea mollis afforded two new brominated arginine-derived alkaloids, subereamines A (1) and B (2), a new brominated phenolic compound, subereaphenol D (3), and the known compounds dichloroverongiaquinol (4), aerothionin (5), and purealdin L (6). The structures of the isolated compounds were assigned using one- and two-dimensional NMR spectra and HRFABMS data. The absolute configurations of subereamines A (1) and B (2) were determined by acid hydrolysis followed by chiral-phase LC-MS. The antimicrobial and antioxidant activities of the isolated compounds have been evaluated. Dichloroverongiaquinol and subereaphenol D displayed significant antimicrobial activity. Using the DPPH TLC autographic rapid screen for free radical scavenging effects, subereaphenol D displayed a significant antioxidant effect. In addition, the cytotoxic activities of the isolated compounds were investigated.

Prostrate cancer constitutes the second leading cause of cancer deaths in men in United States. Eunicellin-based diterpenoids are important bioactive marine natural products isolated from corals of alcyonaria species. The bioactivities of eunicellin diterpenes were correlated with their chemical structures. Recently eunicellin diterpenes from the Red Sea soft coral Cladiella pachyclados showed significant anti-migratory and anti-invasive activities against prostate cancer in wound-healing and Cultrex(®) invasion models. These results encouraged the semisynthetic and 3D-QSAR studies of this unique marine natural product class as possible hits for the control of metastatic prostate cancer. Ten new semisynthetic analogues of cladiellisin (1) were prepared. These include C-6 carbamoylation and ∆(11-17) epoxidation. Carbamate analogues of 1 showed potent anti-migratory and anti-invasive activities against PC-3 cells. Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) were performed using SYBYL 8.1 program package to create a valid 3D-QSAR model to guide future design of potent eunicellin diterpenes cancer migration inhibitors. Eunicellin-based diterpenes are potential marine natural hits appropriate for optimization as inhibitors of metastatic prostate cancer., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

The marine-derived macrolide latrunculins A and B, from the Red Sea sponge Negombata magnifica, are known to reversibly bind actin monomers, forming 1:1 complex with G-actin, disrupting its polymerization. Latrunculins have remarkable physiological properties and widely used as biochemical markers. Nevertheless, no QSAR studies have been developed for any kind of actin disruptors. In the present study, Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) implemented in the SYBYL software packages were used to develop predictive 3D-QSAR models. Two alignment strategies were used to fit analyzed molecules to a suitable conformational template. By means of the SYBYL multifit alignment function, the best CoMFA and CoMSIA results presented cross-validated correlation coefficient values (q(2)) of 0.621 and 0.659, and non-cross-validated values (r(2)) of 0.938 and 0.965, respectively. Comparable to multifit-derived models, CoMFA and CoMSIA 3D-QSAR models were also derived using a molecular alignment obtained by docking latrunculin derivatives into the ATP active site of actin. In addition to q(2), the predictive ability was validated using external test set of five compounds. The results of this study suggest that the established model has a strong predictive ability and can be prospectively used in the molecular design and action mechanism analysis of this kind of cytotoxic compounds., (2010 Elsevier Masson SAS. All rights reserved.)

Alcyonaria species are among the important marine invertebrate classes that produce a wealth of chemically diverse bioactive diterpenes. Examples of these are the potent microtubule disruptor sarcodictyins and eleutherobin. The genus Cladiella has proven to be a rich source of cytotoxic eunicellin-based diterpenoids. Five new eunicellin diterpenes, pachycladins A-E (1-5), were isolated from the Red Sea soft coral Cladiella pachyclados. The known sclerophytin A Cladiellisin, 3-acetylcladiellisin, 3,6-diacetylcladiellisin, (+)-polyanthelin A, klysimplexin G, klysimplexin E, sclerophytin F methyl ether, (6Z)-cladiellin (cladiella-6Z,11(17)-dien-3-ol), sclerophytin B, and patagonicol were also identified. The structures of the isolated compounds were elucidated by extensive interpretation of their spectroscopic data. These compounds were evaluated for their ability to inhibit growth, proliferation, invasion, and migration of the prostate cancer cells PC-3. Some of the new metabolites exhibited significant anti-invasive activity.

The microfilament cytoskeleton protein actin plays an important role in cell biology and affects cytokinesis, morphogenesis, and cell migration. These functions usually fail and become abnormal in cancer cells. The marine-derived macrolides latrunculins A and B, from the Red Sea sponge Negombata magnifica, are known to reversibly bind actin monomers, forming 1:1 stoichiometric complexes with G-actin, disrupting its polymerization. To identify novel therapeutic agents for effective treatment of metastatic breast cancer, several semisynthetic derivatives of latrunculin A with diverse steric, electrostatic, and hydrogen bond donor and acceptor properties were rationally prepared. Analogues were designed to modulate the binding affinity toward G-actin. Examples of these reactions are esterification, acetylation, and N-alkylation. Semisynthetic latrunculins were then tested for their ability to inhibit pyrene-conjugated actin polymerization, and subsequently assayed for their antiproliferative and anti-invasive properties against MCF7 and MDA-MB-231 cells using MTT and invasion assays, respectively.

Bioassay-guided fractionation of the anti-inflammation fractions of the Red Sea sponges Scalarispongia aqabaensis and Callyspongia siphonella yielded two new sterols from chloroform fractions of methanol extracts, namely scalaristerol (5alpha,8alpha-dihydroxycholest-6-en-3beta-ol) (1) from Scalarispongia aqabaensis, and callysterol (ergosta-5,11-dien-3beta-ol) (2) from Callyspongia siphonella. Structure determination was based on extensive NMR studies and mass spectrometry. The antiinflammatory activity of compounds 1 and 2 was assessed using the rat-hind paw edema method and by study of their effect on the release of O2(-) and TXB2 from LPS-activated rat neonatal microglia.

This study reports the isolation of nine new terpenoids (2-10), possessing two novel skeletons, from the Red Sea sponge Callyspongia (=Siphonochalina) siphonella. The identity of these novel skeletons was based on X-ray crystallography and extensive spectral analyses. These compounds were evaluated for their ability to reverse P-glycoprotein (P-gp)-mediated multidrug resistance in human epidermoid cancer cells. Sipholenone E (3) was better than sipholenol A (1), a known P-gp modulator from this sponge, in reversing the P-gp-mediated multidrug resistance. Sipholenol L (6) and siphonellinol D (8) were nearly as active as sipholenol A. On the basis of X-ray crystallographic data and the established identity of 3-7, the structure of sipholenol I (11) is revised. A pharmacophore model of three hydrophobic points and two H-bond acceptors was generated for the active sipholane P-gp modulators.

Ursolic acid (1) and kaempferol (3) are two major constituents of the Mediterranean plant Nerium oleander L. Microbial metabolism of (1) with Aspergillus flavus (ATCC 9170) resulted in the formation of 3-oxo-ursolic acid derivative, ursonic acid (2). On the other hand, Cunninghamella blakesleeana (ATCC 8688A) was able to convert (3) into kaempferol 3-O-beta-D-glucopyranoside (4) as well as the new natural product kaempferol 4'-sulfate (5). Incubation of kaempferol with Mucor ramannianus (ATCC 9628) led to the isolation of one metabolite identified as kaempferol 4'-O-alpha-L-rhamnopyranoside (6). Transformation of kaempferol to the new compound kaempferol 7-O-beta-D-4-O-methylglucopyranoside (7) and herbacetin 8-O-beta-D-glucopyranoside (8) was observed after fermentation with Beauveria bassiana (ATCC 13144). Cytotoxic as well as antioxidant activities of the isolated metabolites were determined.

Reinvestigation of the Red Sea sponge Suberea mollis afforded two new bromotyrosine-derived alkaloids, subereamollines A (1) and B (2), two new brominated phenolic compounds, subereaphenols B (7) and C (9), and the known compounds aerothionin (3), homoaerothionin (4), 11,19-dideoxyfistularin-3 (5), aeroplysinin-1 (6), and aeroplysinin-2 (8). The structure determination of the isolated compounds was assigned using one- and two-dimensional NMR spectra and HRFABMS data. The antimicrobial and antioxidant activities of the isolated compounds have been evaluated. Aeroplysinin-1 displayed significant antimicrobial activity against S. aureus, P. aerugenosa, and K. pneumoniae. The isolated compounds were examined for their antioxidant activity using a 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) solution-based chemical assay. Among the tested compounds, only subereaphenols B and C displayed a significant effect.

The marine-derived macrolides latrunculins A ( 1) and B, from the Red Sea sponge Negombata magnifica, have been found to reversibly bind actin monomers, forming a 1:1 complex with G-actin and disrupting its polymerization. The microfilament protein actin is responsible for several essential functions within the cell such as cytokinesis and cell migration. One of the main binding pharmacophores of 1 to G-actin was identified as the C-17 lactol hydroxyl moiety that binds arginine 210 NH. Latrunculin A-17- O-carbamates 2- 6 were prepared by reaction with the corresponding isocyanates. Latrunculin A ( 1) and carbamates 4- 6 displayed potent anti-invasive activity against the human highly metastatic human prostate cancer PC-3M cells in a Matrigel assay at a concentration range of 50 nM to 1 microM. Latrunculin A ( 1, 500 nM) decreased the disaggregation and cell migration of PC-3M-CT+ spheroids by 3-fold. Carbamates 4 and 5 were 2.5- and 5-fold more active than 1, respectively, in this assay with less actin binding affinity. Latrunculin A ( 1, IC 50 6.7 microM) and its 17- O-[ N-(benzyl)carbamate ( 6, IC 50 29 microM) suppress hypoxia-induced HIF-1 activation in T47D breast tumor cells.

A new latrunculin, oxalatrunculin B (3), was isolated from Red Sea sponge Negombata corticata. Extensive spectroscopic analysis revealed an unprecedented heterocycle in which the rare thiazolidinone ring found in latrunculins was oxidized with three additional oxygens. An actin polymerization inhibition assay agreed with MM-PBSA free energy calculations that 3 binds more weakly than latrunculin B to actin. Significant antifungal and anticancer activity of 3 was found, suggesting an alternate target in addition to actin for latrunculin bioactivity.

Through extensive screening of marine sponge compounds, the authors have found that sipholenol A, a sipholane triterpene isolated from the Red Sea sponge, Callyspongia siphonella, potently reversed multidrug resistance (MDR) in cancer cells that overexpressed P-glycoprotein (P-gp). In experiments, sipholenol A potentiated the cytotoxicity of several P-gp substrate anticancer drugs, including colchicine, vinblastine, and paclitaxel, but not the non-P-gp substrate cisplatin, and significantly reversed the MDR of cancer cells KB-C2 and KB-V1 in a concentration-dependent manner. Furthermore, sipholenol A had no effect on the response to cytotoxic agents in cells lacking P-gp expression or expressing MDR protein 1 or breast cancer resistance protein. Sipholenol A (IC(50) > 50 microM) is not toxic to all the cell lines that were used, regardless of their membrane transporter status. Accumulation and efflux studies with the P-gp substrate [(3)H]-paclitaxel demonstrated that sipholenol A time-dependently increased the intracellular accumulation of [(3)H]-paclitaxel by directly inhibiting P-gp-mediated drug efflux. In addition, sipholenol A did not alter the expression of P-gp after treating KB-C2 and KB-V1 cells for 36 h and 72 h. However, it efficaciously stimulated the activity of ATPase of P-gp and inhibited the photolabeling of this transporter with its transport substrate [(125)I]-iodoarylazidoprazosin. Overall, the present results indicate that sipholenol A efficiently inhibits the function of P-gp through direct interactions, and sipholane triterpenes are a new class of potential reversing agents for treatment of MDR in P-gp-overexpressing tumors.

Nineteen triterpenoids, possessing four different skeletons, have been reported so far from the Red Sea sponge Siphonochalina siphonella. However, no biological activity of these compounds was ever reported. This study describes the isolation of two new triterpenoids, siphonellinol C (3) and sipholenol I (4), along with several known sipholane triterpenoids from the Red Sea sponge Callyspongia (=Siphonochalina) siphonella. Allylic oxidation of the major sipholane triterpenoids, sipholenol A (1) and sipholenone A (2), by selenium dioxide afforded four C-28-oxidized derivatives. Sipholane triterpenoids along with their semisynthetic derivatives were evaluated for their cytotoxicity and effect on reversing P-glycoprotein-mediated MDR to colchicine. Sipholenol A was found to be the most potent, and it increased the sensitivity of resistant KB-C2 cells by 16 times toward colchicine. This is the first report related to reversal of cancer chemotherapy resistance using these triterpenoids.

Biological and chemical investigations of the methanolic crude extract of the Red Sea marine sponge Hyrtios erectus led to the isolation of a novel azepino-indole-type alkaloid named hyrtiazepine (2) and 5-hydroxy-1H-indole-3-carboxylic acid methyl ester (3), together with the known metabolites hyrtiosulawesine (1), 5-hydroxyindole-3-carbaldehyde (4), hyrtiosin A (5), and hyrtiosin B (6). Their structures were elucidated on the basis of mass spectrometry and detailed 2D NMR spectroscopic data. Hyrtiosulawesine (1) displayed a significant antiphospholipase A2 activity with an IC50 value of 14 microM in a fluorometric assay using Crotalus adamanteus venom phospholipase A2.

The soft coral Sarcophyton glaucum is a rich resource of several bioactive cembranoids. Sarcophytol A (1) and sarcophine (2) are cembranoid diterpenes reported from this soft coral and extensively investigated for their cancer chemopreventive properties. This study aimed at investigating the antimetastatic potential of the major cembranoids, sarcophine (2) and 2-epi-16-deoxysarcophine (3), from the Red Sea soft coral S. glaucum. Biocatalytic transformation of 3 using Rhizopus stolonifer ATCC 6227a and Absidia spinosa ATCC 6648 afforded four new metabolites, 5-7 and 9, along with the known 9alpha-hydroxysarcophine (8). Sarcophine, 2-epi-16-deoxysarcophine, and metabolites 5-9 revealed significant antimetastatic activity against the highly metastatic mouse melanoma cell line (B16B15b). Cembranoids demonstrate a great potential for further development as antimetastatic agents.

A gene cluster for the biosynthesis of a new small cyclic peptide, dubbed trichamide, was discovered in the genome of the global, bloom-forming marine cyanobacterium Trichodesmium erythraeum ISM101 because of striking similarities to the previously characterized patellamide biosynthesis cluster. The tri cluster consists of a precursor peptide gene containing the amino acid sequence for mature trichamide, a putative heterocyclization gene, an oxidase, two proteases, and hypothetical genes. Based upon detailed sequence analysis, a structure was predicted for trichamide and confirmed by Fourier transform mass spectrometry. Trichamide consists of 11 amino acids, including two cysteine-derived thiazole groups, and is cyclized by an N C terminal amide bond. As the first natural product reported from T. erythraeum, trichamide shows the power of genome mining in the prediction and discovery of new natural products.